Biodegradation of low-density polyethylene (LDPE) sheet by microalga, Uronema africanum Borge

Plastic (polyethylene) pollution is a severe cause of deterioration of a healthy environment. For example, ingestion of plastics in the animal gut, clogging of water canals and retarded solid waste management. Many conventional methods of polyethylene degradation include UV photooxidation, thermal oxidation, incineration, chemical oxidation and landfill are being practiced. However, these methods are not feasible, costlier and not a complete solution for this global issue. Therefore, plausible, alternative solution for this issue is biodegradation. Microbes such as bacteria, fungi and algae are involved in polyethylene degradation in its natural habitat. Among them, algae were given very less importance. In our present study, a potential microalga, morphologically identified as Uronema africanum Borge, isolated from a waste plastic bag collected from a domestic waste dumping site in a freshwater lake. This microalga was further treated with the LDPE sheet in BBM culture medium. Based on the results obtained from light microscopy, dark field microscopy, GC–MS, FT-IR, SEM and AFM, it was concluded that the microalga has initiated degradation of LDPE sheet within 30 days of incubation. Concurrently, the configuration of corrosions, abrasions, grooves and ridges were found similar with the morphological features of the microalga. For example, the configuration of the radial disc-like attachment structure of the microalga was found corresponding to the abrasions on the surface of LDPE sheet at an average size of 20–30 µm in diameter. Whereas, the configuration of ridges and grooves were found similar with the filamentous nature of the microalga (10–15 µm width). This is a hitherto report on the biodegradation of LDPE sheet by the microalga Uronema africanum Borge.

Isolation of microalga colonized on LDPE sheet. Three different groups of microalgae were targeted for the biodegradation of LDPE sheet (Fig. 4). As a result, the LDPE sheet was colonized by green algae (Chlorophyceae) supplemented in BBM culture medium, whereas other two culture medium have shown very low colonization (Fig. 5). Therefore, the highly colonized green microalga was isolated and maintained in the Algal culture room for further processes (Fig. 6).
Morphological identification of selected microalga. The isolated microalga colonized on LDPE sheet was grown as a filament with uninucleate cells and thus it was classified under the Order Ulotrichales and Family Ulotrichaceae 35 . The cells in the filaments are prominently free and not grouped or clustered with a pointed apical cell. Thus, the genus of the isolated microalga was identified as Uronema. The cells are scarcely constricted at the septum, terminal cell much curved and pointed like a sickle. Chloroplast looks 'C' shaped with only one pyrenoid. The basal cell elongated and shorter than other intercalary cells and enlarged into an attaching disc (Fig. 7). Based on these characteristic features, the isolated green microalga was identified as Uronema africanum Borge 28 . This species has been recorded in Africa, Asia and Europe and differs from Uronema confervicolum by having curved and pointed apical cell. It was reported in Rangoon, Burma by attaching to other algae and plants as epiphytes 29 .
Based on the modern phylogenetic system of classification, the systematic position of the isolated microalgal species U. africanum Borge was retrieved from algaebase database 30  Biodegradation of LDPE sheet by selected microalga. The isolated, pure microalga Uronema africanum Borge was then subjected to treat with LDPE sheet in BBM culture medium. After 30 days of incubation, the LDPE sheet was fully colonized by microalga and visualized like green hair-like structures protrude from the LDPE sheet (Fig. 8).
Optical microscopy on biodegradation of LDPE sheet. The microscopical images of microalga (Uronema africanum) treated LDPE sheet, with different range of magnifications showing, microalgal colonization on the surface of the LDPE sheet by forming a radial disc on its surface (Fig. 9). Hence, the filaments are found attached to the LDPE sheet at an angle of 90° and perpendicular to the sheet. Similarly, the washed LDPE sheets showing the presence of some microalgal filament due to strong attachment on the LDPE sheet by the radial disc. The radial disc like structures are still present on the surface of LDPE sheet and some washed regions showing erosions, abrasions, grooves and ridges (Fig. 10). The erosion was formed due to the radial disc formation, whereas grooves and ridges followed by abrasions was formed by the filaments of the microalga (Fig. 11).  www.nature.com/scientificreports/ Dark field microscopy on biodegradation of LDPE sheet. The dark field microscopical study clearly illustrates that the erosions were formed by the radial discs, whereas abrasions with ridges and grooves were formed by the filaments of the microalga U. africanum (Fig. 12). Furthermore, the configuration of the groove formed by the microalga correspondingly similar with the filamentous structure of the microalga U. africanum.
Gas chromatography and mass spectrometric analysis of control and microalga treated LDPE sample. Based (Fig. 14). Comparatively, the control (U. africanum culture without LDPE sheet) consists of fatty acid methyl esters. However, the microalgae treated sample (U. africanum with LDPE sheet) consists of large number of hydrocarbons. Therefore, it is considered that the generation of hydrocarbons might be due to the biodegradation of LDPE sheet by the respective microalga Uronema africanum.    19,21,23). In this study, Area roughness average (Sa), Roughness peak height (Sp), and Roughness pit height (Sv) are the most important, and determining factor for the biodeterioration of LDPE sheet surface. Therefore, it very clear that there are huge differences in both the surface topography roughness and voltage deflection between the control (microalgae untreated LDPE sheet), and sample (U. africanum treated LDPE sheet). It is found that the Sa (Roughness average), Sp (Roughness peak height), and Sv (Roughness pit height) were higher in the U. africanum treated LDPE sheet (sample), than the microalga untreated LDPE sheet (control) in all the three 5 µm 2 ( Formation of the radial disc-like structure is one of the unique characteristic feature of the microalga Uronema africanum to tightly hold the substrate. In our present study, the same radial disc-like structure found on the www.nature.com/scientificreports/ surface of microalga treated LDPE sheet which is optically seen by microscopical studies. Therefore, the overall microscopical studies resulted that, the dark field microscopy, optical microscopy, scanning electron microscopy and atomic force microscopy have shown the configuration of the radial disc-like structure were correspondingly equal in size at an average diameter of approximately 20-30 µm (Fig. 30). Similar kind of results were obtained for ridges and grooves also, which was configured by the microalgal filaments with an average size of 10-15 µm in width (Fig. 31).

Discussion
The biodegradation of LDPE was reported in fungi, bacteria, and some actinomycetes (Streptomycetaceae). Very least works were reported on the biodegradation of LDPE by photosynthetic algae. Biodegradation of polyethylene by microalgae is an ecofriendly and cost-effectively viable option rather than conventional methods of degradation 22 . Commonly, Oscillatoria, Phormidium, Lyngbya, Nostoc, Spirulina, Hydrocoleum, Chlorella, Pithophora, Stigeoclonium tenue, Anomoeoneis and Nitzschia were reported to colonize the polyethylene bags in aquatic environment 31,32 . Since, the cyanobacteria are adhered strongly to the surface of polyethylene and were not removable by water jet 33 . Similarly, microalgal species includes 10 genera, 7 orders and 9 families were enumerated from the different polyethylene degrading sites of Kota, Rajasthan 24 . Whereas, in this present study, a green microalga, morphologically identified as Uronema africanum Borge was found colonized predominantly on the surface of the polyethylene bag collected from Kallukuttai Lake, near Taramani Railway Station, Chennai. The morphological features of the isolated microalga U. africanum was found similar with the morphological description given for U. africanum by Prasad and Misra 28,34 . In our previous study, a cyanobacterium Dolichospermum spiroides was found to be colonized on the surface of the LDPE sheets 22 . But in this case, a green microalga U. africanum was responsible for the colonization of dumped LDPE sheets. Wherefore, such microalga was isolated to study the biological degradation of LDPE sheets. When the LDPE sheets were treated with U. africanum for 30 days of incubation, the LDPE sheets were completely colonized by the alga and green hair-like structures were seen nakedly protrude from the surface of the LDPE sheets. The U. africanum is a green filamentous alga, and hence the filaments were found attached perpendicular on the surface of LDPE sheet based on the light and dark-field microscopical studies. Concurrently, occurrence of erosions, abrasions, grooves and ridges on the treated LDPE sheets were clearly seen. And their configurations were optically corresponding to the radial disc attachment of the basal cell and filaments of the alga.
Gas chromatography and mass spectrometric analysis (GC-MS) and Fourier transform infra-red spectrometry (FT-IR) are the determining factor for biodegradation of polyethylene 35 . When polyethylene was biologically treated with Klebsiella pneumoniae CH001, generation of saturated fatty acids and carboxylic acids were confirmed by both GC-MS and FT-IR analysis respectively 36,37 . Similarly, most prominent structural changes were observed by FT-IR while LDPE degradation by a fungus Aspergillus clavatus 38 . In case of cyanobacteria, it www.nature.com/scientificreports/ may enhance the surface hydrophilicity of the polyethylene by forming additional carbonyl groups which can be further utilized by other microbes 39,40 . However, in our case, the GC-MS results clearly indicates the presence of hydrocarbons in the U. africanum treated sample (supernatant) when compared to control (microalga untreated supernatant). Simultaneously, from FT-IR results, functional groups such as carboxylic acids, esters, nitro compounds, and amino groups were determined in the sample (U. africanum treated LDPE sheet), but no such functional groups were found in the control (microalga untreated LDPE sheet). Therefore, our results clearly illustrate the biodeterioration of LDPE sheet by microalga U. africanum and found familiar with the results discussed by Vimala and Mathew 37 , and Awasthi et al. 36 .
Based on SEM analysis, microbes isolated from the forest soil and automobile wash-out sludge were found to degrade LDPE by forming cavities on its surface 41 . In our former study, three different microalgae including cyanobacteria, green alga, and diatom were involved to study the biodegradation of LDPE sheet. And among them, the cyanobacterium Dolichospermum spiroides alone forms a cavity on the surface of the treated LDPE sheet based on SEM analysis 22 . In another study, SEM images had shown profuse cracks on the LDPE surface by the adherence of the algae 21 . Similarly, by SEM analysis, erosion, pit formation, and cavities were clearly visible on the surface of the cyanobacteria (Phormidium lucidum and Oscillatoria subbrevis) treated LDPE sheets 33 . Simultaneously, in our study, the occurrence of erosions, abrasions, grooves, and ridges by the microalga U. africanum on the surface of LDPE sheets, were clearly visible by SEM analysis. Area roughness average (Sa), Roughness peak height (Sp), and Roughness pit height (Sv) were determined for both the U. africanum treated LDPE sheet (sample), and microalga untreated LDPE sheet (control) by AFM analysis. As a result, Sa, Sp, and Sv of both surface topography roughness, and voltage deflection were found higher in sample (U. africanum treated LDPE sheet) than the control (microalga untreated LDPE sheet). In adding to this, the configuration of abrasions found similar in size with the radial disc-like structure of the algae (20-30 µm). Simultaneously, the grooves and ridges also found similar to the filamentous nature of the algae (10-15 µm).
The biodegradation rate of polyethylene even after prolonged exposure up to 32 years in soil microbial consortia was found very low 42,43 . In another study, approximately 12 months incubation of LDPE sheet with microbes increase surface roughness when compared with the control, causing deterioration. Aspergillus clavatus treated LDPE sheet for 90 days of incubation in aqueous medium, had shown fractures, erosion and grooves without any prior treatment of the LDPE sheet 38 . Whereas, 126 days were taken for forming small cavities, and pits on the surface of LDPE sheet by the fungal mycelium of A. niger 44 . Weight loss of thermally treated HDPE   38,44 . Azotobacter sp. reported to degrade polyethylene within 45 days of incubation 41 . Cyanobacteria, Phormidium lucidum and Oscillatoria subbrevis have been reported to degrade polyethylene within 6 weeks of incubation 33 . Based on our present study, the microalga Uronema africanum initiated polyethylene (LDPE) degradation within 30 days of incubation.
Therefore, based on the results obtained from our study, this is a hitherto report on the biodegradation of LDPE sheet by photosynthetic, and filamentous microalga Uronema africanum Borge.

Materials and methods
Collection of samples. Dumped waste carry bag sample and water sample were collected from the Kallukuttai lake, nearby Taramani Railway Station, Chennai, Tamil Nadu, India on 28th December 2018. The samples were collected in a sterile polyethylene bags and brought to the laboratory immediately for further processes.
About 250 ml conical flasks were used with 100 ml of culture medium followed by supplementation of 10 numbers of LDPE (low density polyethylene) sheets of 1 cm 2 . The LDPE sheets were supplemented after autoclaving of the culture medium at 15 psi for 15 min. About an inoculation loop full of samples from the collected waste carry bags were inoculated and incubated under light illumination for 12:12 h of light and dark conditions at algal culture room (25 °C). The most common microalga colonizing the LDPE sheet was isolated, sub-cultured and maintained in the algal culture room based on streak plate method.

Biodegradation of LDPE sheet by selected microalga.
The microalga was selected based on the colonization on the surface of the LDPE sheet. Furthermore, the selected and isolated pure microalga was subjected to treat LDPE sheets for its biodegradation studies. In an autoclaved 100 ml of BBM culture medium in a 250 ml conical flask, 10 numbers of 1 cm 2 LDPE sheets were added and the pure selected microalga was inoculated (1% inoculum) and incubated under 12:12 h of light and dark condition in an algal culture room. After 30 days of incubation, the LDPE sheets were taken out from the culture flasks and analyzed for its biodegradation by microalga.
Optical microscopy on biodegradation of LDPE sheet. For optical microscopical study on biodegradation of LDPE sheet by the selected microalga; sophisticated, binocular, compound microscope was employed (Lawrence and Mayo). The image was focused at 10 ×, 40 × and 100 × (oil immersion) magnification and captured using an inbuilt camera along with the microscope using Scopeimage 9.0 software.

Gas chromatography and mass spectrometric analysis of control and microalga treated LDPE sheet sample.
After microalgal treatment of LDPE sheet, the culture medium was centrifuged at 5000 rpm for 10 min. and the supernatant was evaporated and extracted with methanol, followed by GC-MS analysis. Microalgal culture without LDPE treatment was used as a negative control. The gas chromatography was performed by using Perkin Elmer Clarus 680 instrument equipped with fused silica column with Elite-5MS (30 m × 0.25 mm ID × 250 µm df). The mass spectrometry was done by using Perkin Elmer Clarus 600 (EI), the conditions were 240 °C for transfer line temperature the same temperature was followed as ion source temperature. The contents were determined by GC-MS NIST (2008).

Fourier transform infrared spectroscopy (FT-IR) analysis of microalga treated LDPE
sheet. The microalga treated LDPE sheets were analyzed by Infra-Red spectrometer (Bruker, INVENIO R).
The FT-IR spectrum of microalga treated LDPE sheet was obtained as a percentage of transmission ranged from 400 to 4000 cm −1 . The results of microalga treated LDPE sheet was compared with the results obtained from the untreated LDPE sheet (control).

Scanning electron microscopical (SEM) analysis of microalga treated LDPE sheet.
The microalga treated LDPE sheet was subjected to analyze under scanning electron microscopy (SEM). Prior to SEM analysis, the sample could ionize by ion sputter on a metal stub for 20 min. After gold coating, the samples were kept under vacuum to view microscopically by a desktop scanning electron microscope (SEM) (Phenom World Pro) and photographed. For control sample, a fresh and untreated LDPE sheet was employed.

Atomic force microscopical (AFM) analysis of microalga treated LDPE sheet.
To study the surface erosion and biodegradation of LDPE sheet by biological treatment of the selected microalga, atomic force microscopy was employed. Atomic scope microscope Nanosurf Easyscan 2, head type EZ2-AFM with scan head 10-06-176.hed was used. Cantilever type: ContAI-G was used with static force as operating mode and Air was the measurement environment. The Z-controller was set at setpoint: 20 nV, with I-gain: 1000 and P-gain: 10,000. The AFM was controlled using the software version 3.0.1.16 and firmware version 3.1.3.0. The images were taken at 25 µm 2 , 10 µm 2 and 5 µm 2 sizes and analyzed with different mode by software Nanosurf easyscan 2 version 3.8.6.3. A fresh untreated LDPE sheet was used as control. www.nature.com/scientificreports/

Conclusion
In our study, we have isolated a microalga Uronema africanum Borge found colonized on the surface of dumped waste plastic carry bags from highly urbanized freshwater lake. The isolated microalga was subjected to biodegradation of LDPE sheets. Based on the light microscopy, dark field microscopy, GC-MS, FT-IR, SEM and AFM results, it was found that the microalga can initiate degradation of low-density polyethylene in 30 days of incubation. Intriguingly, the configuration of the radial disc-like attachment structure of the microalga was found corresponding to the abrasions on the surface of LDPE sheet at an average size of 20-30 µm in diameter. Whereas, the configuration of ridges and grooves were found similar with the filamentous nature of the microalga (10-15 µm width). Therefore, this is a hitherto report on the biodegradation of LDPE sheet by the microalga Uronema africanum Borge.